This program explores roles of reactive oxygen species (ROS) as specific signaling molecules in B and T lymphocytes through genetic manipulation of the Nox/Duox family of NADPH oxidases. These enzymes are membrane flavocytochromes that catalyze NADPH-dependent reduction of molecular oxygen to generate superoxide and/or hydrogen peroxide. Phagocytes produce large amounts of ROS in response to infectious or inflammatory stimuli through the prototypic NADPH oxidase (Nox) containing gp91phox (Nox2). Recent discovery of multiple homologues of gp91phox (Nox1, Nox3-5, Duox1, Duox2) has opened studies on possible roles of Nox-derived ROS in non-phagocytic cells. Our studies of the functions of Nox family members in lymphocytes provide opportunities to establish distinct roles of deliberate ROS generation in adaptive immune responses to diverse pathogens as well as in autoimmunity or immunodeficiencies. Although originally understood as an anti-bacterial mechanism employed by phagocytes, our research revealed that ROS intentionally generated by several NADPH oxidase family members play specific signaling roles in B cell receptor (BCR)-stimulated B cells and T cell receptor (TCR)-stimulated T cells. We showed that TCR stimulation induces three kinetically distinct ROS generation phases in vitro. Early hydrogen peroxide generation comes from Duox1, activated downstream of inositol 1,4,5 triphosphate receptor 1 and calcium signals; one of the later responses comes from Nox2, activated downstream of the Fas receptor. In 2015, we expanded our efforts characterizing NADPH oxidase-derived redox signals in stimulated B lymphocytes in studies exploring distinct roles of two oxidases: Nox2 and Duox1. Nox2 is the prevalent oxidase detected in human and mouse B cells. Based on differences in BCR-stimulated responses from wild type (WT) and Nox2-deficient mice, it appears to account for most of extracellular hydrogen peroxide detected in the vicinity of the BCR in freshly isolated murine CD19+ splenic B cells. In contrast, we found that 3-day ex vivo treatment of isolated splenic B cells with interleukin-4 (IL-4) in combination with anti-IgM caused a >7-fold induction of Duox1. Interestingly, under these conditions hydrogen peroxide release from WT and Nox2-/- splenic B cells was comparable, whereas ROS release from Duox1-/- cells was greatly reduced. Use of a cell-permeable superoxide-sensitive probe (CellRox) indicated that Nox2 is also activated 24 hours post-stimulation with both IL-4 and anti-IgM. Both WT and Duox-/- co-stimulated splenic B cells produced similar CellRox signals, whereas Nox2-/- cells exhibited reduced signals. Thus, both oxidases show late-phase responses to IL-4/BCR co-stimulation but produce different types of ROS (hydrogen peroxide versus superoxide) within distinct cellular compartments. Activation of the each oxidase under the influence of IL-4 was manifested in distinct downstream functional responses: stimulated Duox1-/- cells exhibited enhanced proliferation relative to WT or Nox2-/- cells (3-4 days later). The effects of Duox1 deficiency on stimulated cell proliferation could be mimicked by treatment of stimulated cells with the hydrogen peroxide scavenger, catalase. Co-stimulation of Nox2-/- cells more readily induced apoptosis and resulted in diminished production of IgM, IgG1 and IgG2a relative to WT or Duox1-/- cells. Taken together, these results indicate that each ROS generator, Duox1 and Nox2, regulates distinct functions in primary CD19+ B cell responses, particularly under the influence of IL-4. Whole animal immunization and infection experiments are necessary to further understand their importance as immune regulators.